Retroviral Vectors and Retroviral Packaging Cell Lines
A retrovirus is an RNA viral molecule encapsulated in a viral protein envelope which infects a host cell, reverse transcribes its RNA molecule into DNA, and integrates its genome stably into the host cell's genome. Systems for packaging retroviral vectors into viral particles to form virions have been developed to facilitate the transfer of exogenous genes into target cells for the purpose of gene therapy. A retroviral vector is DNA or RNA that has been modified to serve as a vector for recombinant DNA. Retroviral vectors can transfer genes into a wide variety of cell types from many different species.
The retroviral particles required to carry out retroviral-mediated gene transfer are produced by packaging cells. Packaging cell lines synthesize all retroviral proteins required for assembly of non-self-replicating infectious retrovirus, and are designed so as to not produce any replication-competent virus. Packaging cells become producer cells when they are transfected with a retroviral vector carrying the gene of interest. Producer cells “package” a retroviral vector into viral particles, thereby producing virions which can be used to transfer the gene of interest to target cells.
The three retroviral genes required to make functional viral particles are (i) gag (encoding internal structure proteins); (ii) pol (encoding RNA dependent DNA polymerase, and protease and integrase proteins); and (iii) env (encoding viral envelope protein). In addition to these three genes, a wild-type retroviral genome usually contains two long terminal repeat sequences (LTRs) and a packaging sequence Ψ sequence). The two LTRs, one located at the 5′ end and the other at the 3′ end of the viral nucleic acid, are necessary for reverse transcription of the viral RNA sequence into DNA and stable integration of the viral genome into the host cell's DNA. The Ψ sequence is required for packaging of viral RNA into the viral particles. Without a nucleic acid sequence containing a Ψ sequence, cells expressing the viral genes gag, pol and env will only produce empty viral particles.
A safety feature of self-replication-deficient retroviral packaging cell lines is that the viral genes required for producing viral particles are placed onto two separate plasmids, with the gag and pol sequences on one plasmid and env sequence on another. Neither plasmid contains the Ψ sequence or a 3′ LTR. When packaging cells are transfected with a viral vector comprising the gene to be transferred, a Ψ sequence and a 3′ LTR, virions carrying the gene of interest are produced. In this system, infectious self-replicating wild-type virus can only be produced if three separate and highly improbable recombination events result in all the required viral genomic sequences (i.e., gag, pol, env and Ψ sequence) coming together in one element.
Packaging cells can express the viral gag, pol and env genes either transiently from separate unintegrated plasmids or stably from these viral genes which have been integrated into the packaging cell's genome. Stable packaging cell lines are advantageous because they can reliably produce the large quantities of virions required for effective retroviral-mediated gene therapy protocols (Bank et al., U.S. Pat. No. 6,372,502 and Markowitz, 1988b).
Challenges to Effective Retroviral-Mediated Gene Therapy
Some cell types are not efficiently transduced by viral particles produced by currently available packaging cells. In order for a viral particle to enter a target cell, the target cell must express a receptor which recognizes the viral envelope protein. Amphotropic retroviruses can infect a wide range of cell types, and therefore, packaging cells expressing amphotropic retroviral genes have been developed and are commonly used (Cone, 1984; Miller, 1986; and Sorge, 1984).
Hematopoietic stem cells (HSCs) are an important target cell type for gene therapy. This cell type can be readily isolated from a patient, transduced ex vivo with a retroviral vector containing the therapeutic gene and then reintroduced into the patient.
One stable retroviral packaging system currently used to produce virions for transducing human hematopoietic stem and progenitors cells expresses the Moloney Leukemia Virus (MLV) gag and pol genes and the GLVR-1 or GLVR-2 viral envelope gene. The GLVR-1 and GLVR-2 retroviral envelope proteins are known to target amphotropic receptors (Markowitz, 1988b; Ward, 1994; and Ward, 1996). Viral particles produced by this type of stable amphotropic packaging system have poor transduction efficiency of HSCs because these target cells express low levels of amphotropic envelope protein receptors on their cell membranes (Orlic, 1996; Kaubisch, 1999; and Kavanaugh, 1994). In addition, amphotropic stable packaging cell lines generally are unable to produce the requisite titer (106 or greater viral particles per ml) necessary to compensate for the low levels of receptor expression on HSCs. Researchers have reported that amphotropic viruses pseudotyped with other viral envelopes, such as the vesticular stomatitis G protein (VSV-G), can improve transduction efficiency. The VSV-G envelope protein enables effective targeting of a variety of cells, including HSCs. Higher viral titers can be achieved using VSV-G envelope protein pseudotyped viral particles because these viral particle supernatants can be concentrated by ultracentrifugation (Burns, 1993). One limitation of this system is that expression of the VSV-G protein is toxic to the packaging cells. Consequently, only transient or inducible packaging lines are feasible using this viral envelope protein.
An alternative to the VSV-G envelope protein is the feline endogenous virus envelope (RD114). RD114 pseudotyped viral particles are able to efficiently target human HSCs and can be concentrated by ultracentrifugation (Porter, 1996; and Gatlin, 2001). A RD114 pseudotyped virion has been shown to transfer the green fluorescent protein (GFP) gene into human HSCs at high levels (Kelly, 2000). Virions used in this study were produced by a transient retroviral packaging system in which unintegrated expression plasmids for gag and pol genes, and the RD114 envelope protein gene were transiently transfected into 293T cells (derived from human embryonic kidney cells) Transient packaging systems, however, can not produce the large quantities of virions required for human clinical trials.
The FLYRD18 packaging cell line is a stable packaging cell line that produces RD114 pseudotyped virions. However, the use of this cell line for producing virions for transducing HSCs is limited because the viral supernatant produced by these packaging cells causes phenotypic changes and loss of primitive repopulating cells (Kelly, 2000).
For successful transduction of HSCs and other target cell types for gene therapy in humans, a viral packaging cell line must (i) produce large quantities of retroviral particle supernatants; (ii) produce viral particles that efficiently transduce the target cell; and (iii) only produce replication-defective viral particles. To date, there remains a need for such cell lines.